Vitamin b12-active composition and process of preparing same



United States PatC nfO VITAMIN Biz-ACTIVE COMPOSITION AND PROCESS oEPREPARlNG SAME Edward L. Rickes, Rahway, N. J., and Thomas R. Wood,

Hockessin, DeL, assignors to Merck & Co., Inc., Rahway, N. J., a corporation of New Jersey No Drawing. Application December 8, 1952, Serial No. 324,834

12 Claims. (Cl. 167-81) erty of promoting the growth of the microorganism 4 Lactobacillus lactzs Dorner (LLD), and to methods for producing such vitamin Biz-active, APF-active and LLD- active materials utilizing selected strains of microorganisms belonging to the subphylum Fungi. These vitamin Biz-active concentrates are valuable as feed supplements and for the treatment of nutritional diseases.

This application is a continuation-in-part of our copending applications Serial No. 38,175, filed July 10, 1948, now abandoned, Serial No. 110,222, filed August 4, 1949, now Patent No. 2,695,862, and Serial No. 146,404, filed February 25, 1950, which applications are,

No. 20,356, filed April 10, 1948, now abandoned.

It has long been known that the usual animal feeds and, in particular, those used for poultry, composed primarily of plant materials which contain edible vegetable protein material, such as cereal grains, soy bean meal, alfalfa, and the like, and adequate with respect to their content of anime acids, minerals, and vitamins of known chemical structure, are deficient in a factor or factors required for maximum rate of growth and adequate reproduction. This factor is chara'cterizedby a pronounced tendency to be'stored in the tissues and to be carried through the egg to the chick. Hens must be fed rations containing this factor in order to maintain adequacy of egg hatchability and viability of the chicks hatched. Chicks must receive the substance-either directly in their feed or indirectly through the egg from the hen in order to grow at optimum rate and to avoid excessive mortality. It is obvious that the factor is of great economic importance to the poultryman.

In commercial practice good quality practical poultry feeds contain this so-called animal protein factor (which will be referred to in this application as APF) in the form of meat by-products, liver meal, fish meal, fish solubles, and the like. These materials are in relatively short supply. Both meat by-products and liver meal are expensive and add considerably to the cost of poultry raising. These substances are also open to the objection that they contain comparatively small and varying amounts of th'e'APF. It has therefore been desired to find a more concentrated, standardized, and less expensive source of this important dietary constituent.

We have now discovered that APP concentrates, that is products containing a relatively high concentration of APF active constituents and adapted for'the enrichment of animal feeds deficient in the animal protein factor, can be prepared by fermenting aqueous nutrient medium by means of selected strains of microorganisms belonging to the subphylum Fungi and recovering APP active materials from the fermentation broths thus obtained. The strains of Fungi which we employ in preparing APP-active concentrates are those which are capable of producing fermentation broths containing the LLD growth factor required for the growth of the microorganism, Lactobacillzls lactis Dorner, and which possess the additional property of synthesizing vitamin B12 and/or vitamin B12-1ike compounds. 4

Whena vitamin Biz-producing strain of Fungi is. used to ferment an aqueous :nutrient. medium and the ferin turn, continuations-in-part of our application Serial Cir pure vitamin B12 is obtained therefrom. Vitamin B12 is a new chemical compound fully characterized later in this specification, and disclosed and claimed in our Patent No. 2,563,794, which issued on August 7, 1951. Vitamin B12 is capable of promoting the growth of Lactobacillus lactis Dorner and possesses marked and effective action in the therapeutic treatment of Addisonian pernicious anemia and other macrocytic anemias. We have discovered that, in an otherwise APF-deficient chick feed, a level of approximately 0.000003% of vitamin B12 (i. e., 30 micrograms of vitamin B12 per kg. of feed) is capable of producing a satisfactory and apparently maximal effect on the growth rate of chicks.

By vitamin B12-like compounds are meant the red crystalline compounds (not vitamin B12) which are obtained when a fermented broth containing them is subjected to the purification treatment utilized in the preparation ofpure vitamin B12 (as described in columns 7 and 8 of this application) but omitting the countercurrent distribution procedure employed as the final step in this purification operation. These vitamin Biz-like compounds can be characterized by the fact that they are readily convertible to the pure vitamin B12 per se by treatment with cyanide ion, as disclosed in U. S. Patent No. 2,530,416, which issued on November 11, 1950. Vitamin B12 and vitamin Biz-like compounds can be referred to by the expression vitamin Biz-active compounds. These vitamin Biz-active materials, i. e., vitamin B12 and the vitamin Bi2-like compounds, possess APF- activity and can be used as feed supplements for rendering APP-deficient feeds dietetically adequate. They also possess marked and effective action in the treatment of macrocytic anemias.

The strains of Fungi which produce LLD-active substances (and which are potential producers of APF, vitamin B12 and vitamin Biz-like substances) are selected by testing their fermentation broths for the presence (or absence) of the LLD growth factor. The assay of these fermentation broths for LLD content is carried out by utilizing the growth response to the LLD factor of the microorganism, Lactobacillus lactis Dorner, A. T. C. C.,

10,697, which is available in the American Type Culture Collection. Hencefore in this application, this microorganism will be referred to by the abbreviated name, L. lactis. The fermentation of an aqueous nutrient medium by L. lactis results in the production of an acid; the amount of acid produced has been found to be a satisfactory measure of the growth of the microorganism.

L. lactis has been reported to require two growth factors, T. I. and LLD. The basal medium presently described, which eliminates the requirement for the T. J.

' factor, does not contain the LLD factor and, alone, does so merited broth is SUbJCCiCd' to a purification operation,

DL isoleucine mg 200 DL alpha-alanine mg 200 DL aspartic acid mg. 200 DL valine l mg 200 DL methionine mg 200 DL glutamic acid mg 200 DL threonine mg 200 DL serine mg 200 DL phenylalanine mg 200 DL leucine mg 200 L histidine mg 200 'DL tryptophane rng 400 L-arginine mg 200 L-lysine' mg Aminoacetic acid mg 200 L cystine mg 200 'DL norleucine mg 200 L tyrosine mg 200 Dextrosegm 10 Sodium acetate gm 6 Fumaric acid gm 0.5 Sodium ethyloxalacetate gm 0.5 Riboflavin rncg 200 Calcium pantothenate mcg 200 Thiamin HCl mcg 200 hNicotinic acid mcg; 200

not support growth of L. lactis.

The composition of the basal medium, double strength,

is as follows:

Water to 500 cc.

The basal medium is prepared by combining the amino acids, then adding dextrose, sodium acetate, fumaric acid,- heating to dissolve, and immediately readjusting to pH 7. The sodium ethyloxalacetate and vitamins are then added, dissolved, and the solutions again adjusted to. pH 7. Finally, the salts, folic acid and casein hydrolyzate are added, dissolved, and the pH adjusted to 6.6.

As noted above, this basal medium does not support the growth of L. lactis. When a small amount of LLD active substance, as for examplepure vitamin B12, is added to this basal medium, the medium thus supplemented does, however, support the growth. of L. lactis, and the extent of growth is a direct function of the amount of LLD growth factor added. The LLD; factorcontent of the substance added to the medium can therefore be measured by fermenting the supplementedmedi-um by means of L. lactis under controlled conditions of time and temperature and then titrating the acid produced-during the fermentation. The LLD factor content of substances thus assayed is expressed in terms ofthe arbitrarily assigned LLD unit; pure crystalline vitamin B12 contains 11X LLD units per milligram.

The relationship between the LLD units added to the basal medium and the acid produced: by fermentation of the medium by L. lactis is expressed by meansof a standard curve which is plotted from values obtained using,

pure crystaline vitamin B12 as a standardized source. of the LLD factor. These values are obtained as follows: Stock cultures of L. lactisare maintainedon a growth medium which consists of: 1% Difco. yeast extract, 0.02% tomato juice serum, 1% anhydrous dextrose, and 1.5% agar. An inoculum is prepared using-as a medium the basal medium previously described. to which has been added an amount of vitamin B12 equivalent to,

l LLD unit per cc. of medium. The inoculum cells thus obtainedare washed with sterile distilled water and.-dil-. uted to form a standardized suspension of L. lactis which reads between 90% and 95% light transmission on .the Evelyn photometer with a 520 a filten.

A stock solution of vitamin B12 is diluted so. that the solution contains exactly 0.8 LLD units: per cc., (i. e., 0.000073 microgram of vitamin B12 per cc.) and 0.0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0 and. 2.0, LLD units are added to duplicate series of tubes. The volume of solution in each tube is adjusted to 2.5 cc.v by addition. of water, 2.5 cc. of the basal medium is added to each tube, and the. tubes are plugged and sterilized by heating at 120 C. for 13 minutes. After cooling to-room tem perature, the tubes are inoculated with one drop of a thestandardized suspension of L. lactis prepared-as described above and then incubated at 37% C. for 40 hours. After incubation, the tubes are titrated directly with 0.05 N sodium hydroxide to the blue-green endpoint-of bromthymol blue. Typical titration values forthe standard series listed above are: 1.5, 2.0, 2.7, 4.3,5.7, 6.6, 7.5, 7.9, 8.0 and 8.4 respectively, in terms of the milliliters of 0.05 N sodium hydroxide required toneutralize the acid produced per culture of L. lactis. The standard curve is then obtained by plotting each of these titration values against the value for the LLD units originally added to the corresponding tube prior to fermentation.

In order to select an LLD-activity. producing strainv of Fungi, a culture of the microorganism under investigation is diluted, plated out on a solid nutrient medium, and incubated to produce a considerable numberofsin gle cell colonies. Individual colonies, picked for inoculum development, are then grown in a liquid medium,

supplemented with cobalt nitrate at a concentration of 0.1 part of cobalt per million parts of .medium;undersubmerged conditions in shake flasks. A sample offermented broth from each shake flashv is. then". assayed for LLD activity.

A preliminary experiment is carried out in order to determine fl) whetherthe broth has-any- LLD activity; and (2) the approximate value of this LLD activity. The fermented broth from each shake flask is diluted with approximately 500 parts of water (for a broth containing LLD units per ce., .the diluted broth would thus contain approximately 0.2 LLD unit per 00.), and 2.5 CC.'Of the resulting-solution is mixed with 2.5 cc. of the LLD assay-basal medium; The tubes'are then sterilized, inoculated, and incubated in the same manner employed in obtaining thevalues used in plotting-the stand ard curve; after incubation, the contents of the tubes are titrated with 0.05 N sodium hydroxide to the blue-green endpoint of bromthymol blue. The-total .LLD units contained in the tube priortto the-fermentation can then be read from the standard curve at the point corresponding to the above titration value. The.above approximate test is sufficient to determine whether or not a given strain of. Fungi is-.-capab1e-of producing;LLD-activc substances.

An exact value for. the LLD content of these Fungi fermentation broths, which is indicative of the capacity possessed. by the particular strain--for"producing. vitamin B1: or other APF active substances; is.-determined as follows: A-sample of the fermentationubroth from each shake flask isv diluted, on: thebasis ofthe approximate LLD activity determined as describedabove; so that the solution-contains 0&2 LLD unit per cc., and this solution is added to the assay tubes in 015, 1.0, 1.5, 2.0 and 2.5 cc. amounts. The volume.of-solutionineach tube is then adjusted to a volume of 2.5 cc. bygaddition of water, 2.5 ccof the|basal medium is-added, and the tubes are sterilized, inoculated, incubated, and titrated with 0:05 N sodium hydroxide as prexiously described. Comparison of the. titration values thus-obtainedwitnthe standard curve gives a series ofvalues for the LLD units contained in'cach of the assay'tubesand, by a simple calculation, theprecise LLD: content of the broth taken from the original shake flasks.

It is a feature of the present invention that, instead of isolating thevitamin-Biz, vitaminBiz-like compounds, or. other APP-active substance in pure form from fermentation broths, concentrates of saidbroths can be employed as feed supplements in. amounts depending on the: content of APF activesubstances-as measured by the hereinafter described-chick test. We have discovered that-such concentrates can be preparedtin a commercially practicable way by fermenting; an=aqueous-.nutrient medium with selected strains ofFungi;,. and'then'scparating volatile-constituents from'the resultingfermented broths at-temperatures below those destructive of said APP vitamin substances.- We have'found that the dried residue -from=such filtered=fermented broths can have a po tcncy of aswhigh as about- LLD units per mg. and that concentrates COIlt3iI1l'I1g' 2,0Q0LLD units-per mg. or higher, are readily obtainable. In contrast to this the richest sources of APF-previously available, such as fish solubles and'fish'meal, havcnbeenfound tocontain only about 1-3 LLD units perv mg.;

It should be noted that an= APP-deficient basal-poultry ration is rendered dietetically adequate with respect to APP by adding to said ration- 0.000003% ofvitamin B12, andthat this amountof'vitarnin B12 produces a growth response inchicks equivalent'to that produced by about -1 to 5%=fish--solubles,fish meals, and the like. Thisratio-of vitamin B12 corersponds to a level of about 330,000-LLD. units per kg.,of-feed.- A- concentrate of 2000 LLD units per. mg. potency, such-as that mentioned above, supplies a level-.of-.330, 000-LLD units per kg. .of feed when added in the-proportionof 165 mg. of concentrate per-kg. of feed (i. e., 0.0l65.%

It-is.-known that--various. fermentation solubles, that is, the products obtained by evaporating to-dryness fermentation liquors. produced-in the manufacture of butyl alcohol, grain alcohol, etc.. have been employed as feed supplementsprimarily becauseof'their content of ribofiavinand-other vitamins of known structure, such as biotinnpantothenicacid, etc. There is no indication in the- ;prior art, however, that Fungi fermentation liquors might-contain vitamin substances which would be useful as .feed'supplements for enriching feedsdeficient in animal protein; factor.

We have found that the priorrartzfermentation solubles containonlytsma-ll .amountstofiLLDsactive"substances; for examplegrbutyl fermentationisolublescontain only about 4 LLDiunitsspenmgmand grainndistillers solubles'eontainless than 1 LLD unit permg. Wehavebeen'unable to isolate any vitamin B12 from these materials and none of these prior art fermentation solubles has proved satisfactory for the enrichment of foods deficient in APF.

Since many LLD activity-producing strains of Fungi produce only small or'varying amounts of APF, we ordinarily prefer to prepare APF-activesubsta'nces utilizing those strains of Fungi capable of producing fermentation liquors which, when concentrated and the concentrate added to an APP-deficient chick feed in'an amount not exceeding by weight of said feed and the enriched feed fed to chicks hatched from eggs laid by hens maintained on an APP-deficient diet, will produce a maximal rate of growth in said chicks comparable with that obtained by feeding the chicks said 'APF-deficient chick feed supplemented with 30 micrograms of vitamin B12 per kilogram of feed. Assay of these fermentation residues obtained utilizing said preferred strains of Fungi has established that such fermentation residues contain LLD activity. As noted above, fermentation residues containing 140 LLD units per milligram and higher are frequently secured by evaporating fermentation broths obtained utilizing these preferred strains of Fungi.

The microorganisms which include these LLD-activity and potential APP-producing strains are the Fungi as outlined on page 2 of the book Introduction to Industrial Mycology by Smith and Raistrick, London, Edward Arnold & Co. (1938), that is, the Myxomycetes, Schizomycetes, and Eumycetes. We ordinarily employ selected strains of microorganismsbelonging to the Schizomycetes, particularly certain strains of the genus Bacillus, the genus Streptomyces, the genus Alcaligenes, the genus Pseudomonas, the genus Mycobacterium, and the genus Escherichia. The preferred Eumycetes includes strains of the genus Torula, the genus Eremothecium, and the genus Alternaria. We prefer to utlize strains of Fungi selected from the following species, namely: Streptomyces griseus, Streptomyces albidoflavus, Streptomyces colombiensis nov. sp., Streptomyces roseochromogenus, Streptomyces antibioticus, Streptomyces fradiae, Streptomyces venezulae, Alcaligenes faecalis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mildenbergii, Pseudomonas mucidolens, Pseudomo nas lumichroma, Pseudomonas chloraraphis, Mycobacterium smegmatis, and Alternaria alevaeca, and, in particular, those strains of Streptomyces griseus which, in addition to synthesizing vitamin Big or other APF-active substances also produce one of the antibiotics, streptomycin or grisein.

We wish to emphasize, however, that for any given species of Fungi, it is necessary to select strains which produce fermentation residues having LLD activity and which produce growth stimulation in chicks when used to supplement APP-deficient feeds as outlined in the preceding paragraph and as fully described in the chick test set forth herein below. Our invention is neither lim- 1ted to any species of Fungi, nor does it include every strain of any given species. On the other hand, every strain of Fungi yet tried, which produces fermentation residues containing 4 LLD units per mg. and satisfying the requirement of the chick test, has been satisfactory for producing concentrates adapted for the enrichment of animal feeds deficient in the animal protein factor.

In order to select these preferred strains of Fungi, fermented broths produced in shake flasks by LLD-activity producing strains as determined hereinabove are evaporated to dryness under reduced pressure at temperatures below those destructive of the vitamin content. The fermentation residues thus obtained are assayed for LLD content. Those strains, which produce fermentation residues having an LLD content of at least 4 LLD units per milligram are used to prepare additional quantities of the corresponding fermentation residues.

Various procedures may be employed for preparing these fermentation residues from the fermented broths. For example, the broth may be filtered to remove microorganism cellular debris and other insoluble ,matter and the filtrate concentrated by evaporation followed by spray drying to produce a solid concentrate for the APF chick test. A very convenient method for the preparation of the dried fermentation residue is to freeze dry the whole fermented broth.

If a fermentation residue prepared by the above described methods gives evidence-of toxicity whenfedto chicks, the culture which produced it is notdiscarded,-bu t instead certain methods for removal of toxic; factors, well;-

known to chemists skilled in the incident art,are applied to. the fermented broth and crude fermentation residue. Such methods involve treatment of the fermented broth by heat, or with selective extractants such as .ion exchange resins, solvents, and the like. If none of these'toxin removal methods is successful, the strain under investigation is discarded as being of no interest from a practical standpoint for the production of an APF concentrate.

These fermentation residues which are not toxic to chicks, or from which the toxic factors have been removed, are added to an APP-deficient basal diet and the supplemented diet tested for growth response in chicks. This chick test is carried out using chicks hatched from eggs laid by hens maintained on a diet deficient in APF. Day-old chicks are placed on a basal diet (which does not contain APF) as follows:

Percent Soy bean meal 70.0 Commercial dextrose 7.9 Cellulose 5.0 Wheat germ oil 4.5 Calcium gluconate 2.5 Glycine 2.0 DL-methionine 0.9 C'aCOz 1.5 K2HPO4 1.612 KH2PO4 1.0 NaCl 0.838 MgSOt-7H2O v 0.51 Cal-IP04 2H2O 0.375 Ferric citrate 0.138 MnSOr-4Hz0 0.025 KI 0.004 CIISO4-SHzO 0.0015 ZnClz 0.00125 L-Arginine 0.50 L-Cysteine 0.20 Choline 0.20 Inositol 0.10 p-Aminobenzoic acid 0.03 Niacin 0.01 Ca pantothenate 0.004 Riboflavin 0.002 Thiamin 0.002 Pyridoxine 0.002 Pteroylglutamic acid 0.0004 Menadione 0.0004 Biotin 0.00004 A & D powder 0.10

After 5 days on the above basal diet, four groups of 7 chicks each, balanced in regard to body weight, are selected and each group is given the foregoing basal diet supplement as follows:

(1) No supplement added to basal diet.

(2) 0.0d00003% crystalline vitamin B12 is added to basal iet.

(3) Ten percent by weight of fermentation residue derived from strain of Fungi under consideration is added to basal diet.

(4) The same fermentation residue referred to in item (3) is added to the basal diet in sutficient amount to (provide 330,000 LLD units per kilogram of The average weight of the chicks in each of the four groups fed the indicated diets is determined every 2 or 3 days over a period of approximately 3 weeks. If the chicks, fed either of the diets containingthe fermentation residue, as specified under either (3) or (4) above, show a maximal rate of growth comparable with that obtained in the group of chicks fed diet (2), then that strain of Fungi from which this fermentation residue has been obtained is classified as one of the preferred strains.

As noted hereinabove, we have further discovered that certain of these LLD-activity producing strains of Fungi are capable of synthesizing vitamin B12 and/or vitamin BIZ-like compounds, and the quantity of these substances contained in a fermentation broth can be definitely established by a chemical assay procedure. In view of the fact that vitamin B12 and vitamin Biz-like compounds possess APF activity, this chemical assay procedure provides a reliable and convenient method for selecting strains of Fungi which produce APF active substances;

In order to select a strain of Fungi which produces with l liter of -water. minutes in a mixture of 225 cc. of n-butanol and250 cc. of water and the mixture is filtered to remove the carbon .vitamin B12 Maud/or -vitamin Biz-like compounds, -an .LLD-activity: producingstrain (selected utilizing the-pre- =viously described LLD test), is used to ferment-about' 15 -liters ofan aqueous nutrient medium containing approxi- --mately"'.l--parts per million of cobalt added as cobalt nitrate, said-fermentation being carried out under submerged aerated conditions. The mycelium is removed fromwthe resulting fermented broth, the fermentation liquor is adjusted to a pHof 740, 75 grams of activated carbon (Norit A) -is added and the mixture isslurried for 15 minutes. Theslurry is filtered using a precoat of diatomaceous silica (Supercel) and the carbon iswashed -'The wet carbon is slurried forl5 cake is washed with water and the washings are. added a to the water layer from the original filtrate contained in the graduate. The washing is continued until thereihas accumulated a total of 400 cc. of rich water (combined volume of the water'layer from original filtrate plus washings). If the last few cubic centimeters of wash are still highly colored, the carbon cake is washed with additionaLwater until the color of the wash becomes very light.

This combined Water layer and washings of about 400 cc. .volume is placed in a separatory funnel with 60 percent of its weight of ammonium sulfate and 15 percent of its volume of benzyl alcohol. The mixture is shaken thoroughly and allowedto settle. The'benzyl alcohol layer is separated and dried over sodium sulfate, and then decanted and dried with more sodium sulfate and filtered. -This'first rich benzyl alcohol is placed on a chromatographing column which contains 5 cc. of an activated alumina'saturated with methanol.

The water layer-from the first benzyl alcohol extrac tionis again extracted with 8% of its volume of fresh benzyl alcohol. This second benzyl alcohol extract is dried in two stages over the same sodium sulfate used for drying the firstbenzyl alcohol extract. After drying, the second. benzyl alcoholextract is filtered and addedto the first benzylalcoholextract on the chromatographing column. ,Asmallamount (10 to cc.) of-benZyl-alcohol is used forwashing the flasks and the sodium sulfate on the filter. This-wash .is added to the benzyl alcohol already on the chromatographing column.

After all of the benzyl .alcohol has passed through the column, the column is washed with a mixtureof two volumes of acetone to one of methanol until all of the benzyl alcoholhas been removed from the column and the efiluent is water white. Anhydrous methanol is then added to the'column to develop the. color, the rich methanol cut is collected and four volumes of anhydrous ethyl ether are added'thereto' whereupon a red-coloredprecipi- .tate forms. This j precipitate is recovered by filtering through amediumporosity' fritted glassfilter. The precipitateon the filter is then removed from the filterby dissolving'it in S. cc. of. water saturated with benzyl alcohol which is added dropwise' to the material on the filter.

If the fermentedbroth, prepared utilizing the-strain of Fungi under-consideration, contained vitamin B12, and/ or vitamin'Biz-like compounds, these substances, it has been established, willibe present, in concentrated form, in this aqueous benzyl alcohol solution. The aqueous benzyl alcohol solution is therefore tested, using .a spectrophotometer, for absorptionwithin the range 5200 to 5500A wave length. -If .the aqueous benzyl alcoholsolution shows an absorption maximum at 5200-5500A wave length (such absorption; property being characteristic of vitamin-B12 as well as-of-vitamin Biz-like compounds) then' said solution contains vitamin Biz and/orvitamin Biz-like compounds, andthestrain of Fungi under consideration is:- a producerof :at least one APP-active substancecf-the group consisting-of vitaminBizand vitamin But-like compounds.

Where it.-isdesiredrto=have a quantitative measure of the-total vitamin-B1z and vitaminr Bri-like'material present -in the vbenzyl =1 alcohol solution, "the 'optical 'density at -5500A:waveflengthref said solution is determined on a spectroph0.tometer. compared withastandard solution of vitaminBrz. :The value"thus. obtained.is a measure of the total vitamin. B1 and vitaminLBm-like. compounds.

"The presence of vitaminfBizrper se in this aqueous "benzyl alcohol solution and .the.amount of vitamin B12. .contained' therein arei'determined by, running an 8-plate counter-current .distribution using. .a benzyl alcohol-water system. "The-'-fourth .plateIis used for determining the vitamin B12contentylhe.activity. is. transferred completely into the'S cc. waterllayer onithis .plate' by adding 10 cc. of chloroform-to the 5..cc. benzyl, alcohol layer. Theop- "tical den'sityforthis water. layerisidetermined on.a spectrophotometersat 5500A 'wave-length andthe amount of -vitamin B1z. is calculated as follows:

Optical tlensity'rcadingxcc..water layer-inmate l i i-P Cell thlckness'in em. 6.4X0:29 ,13

No'rn.-'The' figure 6.4 represents the optical density for a one centimeter thickness Ol-solutloncontainlng 1 mg; of vitamin B11 per 00. and 0.29 is the traction otthe total vitamin-Bu present in the fourth plate.

Fromthe above test, 'it is'thus-possible to determine whether-a given strain of Fungi iscapable of synthesizing vitamin-Biz, -as' well asthe amount of vitamin B12.COH- 'tained in the fermentation broth obtained when said strain isxusedto ferment an aqueous nutrient medium. --All vitamin -"B1z-synthesizing strains of Fungi, as deter- 'mine'd by' this test,- produce fermented broths having LLD 'activity and APF active fermentation residues which support. the growth of chicks. We particularly desire to utilize-.these vitaminBiz-synthesizing strains of Fungi for thepreparation-ofrAPF concentrates because it is possible, employing methods ofpurification which we have discovered; to re'finetheactive principle and produce APF concentrates-having:anydesired' activity up to that possessed by pure crystalline vitamin B12- A still' further advantage of the present .discovery lies in the fact thatthe .valuable' vitamin products herein described can be prepared -from waste fermentation liquorsresulting"from"the'production of the antibiotics streptomycin "and *grisein. 'Thestreptomycin and grisein "fermentation *processes involve the fermentation of ma'shesof low 'concentrations'with the result that extremelylarge volumes of fermentation'broth must be disposed offor-ar'elativelysmall.amount of streptomycin or grlsein production. 'Theresiduefrom a large streptomycin "fermentation 'plant may thus constitute many thousands of -=gallons of waste fermentation liquors .per

"Nouseful-product waspreviously known to be present in-these'waste-liquors. In fact, the total amount of vitarnins of known structure actually contained in such liquors is too small to make it'economical to concentrate 'the' liquors for-"the recovery-of said vitamins. These streptomycin fermentation liquors have therefore constituted-a serious and costly waste disposal problem.

iWehavenowmade-the surprising discovery that, by utilizingastrain of Streptomyc-es griset/s which produces both streptomycin and vitamin B12 (or other APF active substance) randaby removingxthestreptomycin from the fermentationbroth by absorption on an ion exchange resin-whosepolar'groups are carboxyl groups, there may be :obtained a waste streptomycin liquor substantially free 'of streptomycin and containing said vitamin B12 (or other 'APF active substance) in a concentration as measured by'the-LLD test of-over-QO LLD units per milliliter. We havedemonstrated that concentrates of high LLD activity can'be:recovered'from this liquor, and that such concentrates can be used for the enrichment of animal feedsdeficient in the animal protein factor. When the .strainofStreptomyces produces vitamin B12 in addition to .streptomycin,=the-wasteliquor (after removal of the streptomycin) -can -be processed, if desired, to produce pure-vitamin-Biz. "Likewise'waste-grisein liquors after recovery --of the grisein antibiotic, may be treated to re cover LlLD-rich--concentrates or 'pure' vitamin B12.

lnrcarrying out:the -presentinvention, we can employ any of the aqueous nutrient mediums ordinarily utilized inthe propagation'of Fungi. 'lhe usual nutrients include *acarbon asource, za nitrogen source, inorganic salts and ugrowlthtfactors:whenrequired. We ordinarily-prefer to supplemcnt the medium with" a :source of cobalt'such as cobalt nitrate. The carbon can be provided by a carbohydrate such as dextrose, maltose, xylose, invert sugar, corn syrup and the like. The nitrogen can be provided by an ammonium salt, amino acids, or proteins, such as soy beans, oats, yeast, yeast extracts, tryptic digest of casein, meat extract, blood meal, protein meat and bone scrap, salmon meaL'fish meals, fish solubles, distillers solubles, and the like; If desired, the Fungi can be propagated using proteins (or amino acids) without any carbohydrate being present in the medium, in which case the proteins (or amino acids) serve as the source of both the carbon and nitrogen required by the microorganism. The propagation is ordinarily carried out in an aqueous medium but a grain bran medium may also be used in place of said aqueous medium.

When an aqueous nutrient medium is used, it is sterilized and then inoculated with a culture of the selected strain of Fungi and the mixture is incubated under submerged conditions and at a temperature appropriate for the specific strain of Fungi employed. The potency (i. e. the LLD activity) of the fermentation broth thus obtained is assayed utilizing the growth response of the microorganism Lactobacillus lactis Dorner as-referred to earlier in this application. The fermentation broth is then evaporated and the fermentation residue is tested for the presence of toxic factors and for APF activity by means of the previously described chick test. If the fermentation residue gives no evidence of toxicity and is APF active, it may be used, without further treatment, to enrich animal feeds deficient in APF.

It is ordinarily preferred, however, to filter the fermentation broth and treat the filtered brothwith an adorbent such as fullers earth or activated charcoal, thereby adsorbing the APF active substances. This adsorbate is dried and can then be used as a feedsupplement. If a feed supplement free of adsorbent is desired, the adsorbate can be eluted with suitable solvents such as aqueous or aqueous alcoholic solutions of pyridine or a-picoline, for example an aqueous butanolsolution of pyridine, and the resulting eluate evaporated to dryness. This procedure effects a partial purification of the APF active constituents and the solid product thus obtained is a concentrated source of growth factors adapted for the enrichment of APF deficient animal feeds.

When a strain of Streptomyces griseus which produces both'streptomycin and APF active substances is used to ferment an aqueous nutrient medium as set forth above, and the fermentation broth evaporated to dryness, there is obtained a fermentation residue which contains some substances relatively toxic to chicks. It is therefore preferred to first recover the streptomycin from the fermentation broth and then to prepare a concentrate having APP-activity and free of chicktoxic substances from the waste liquor thus obtained. The streptomycin is conveniently recovered from the fermentation broth, as described in U. S. Patent 2,541,420, issued February 13, 1951, by adsorption with an ion exchange resin whose polar groups are carboxyl groups. This treatment also removes substances toxic to chicks. We have discovered that these resins offer substantial advantages over all other adsorbents which have been tried since they not only accomplish theadsorption of the streptomycin present in the broth in substantially quantitative yield and in a state of high purity, but also, at the same time, yield an effluent containing substantially all of the vitamin B12 or other APF-active substance present in the original fermentation broth.

The efiluentbroth, from which the streptomycin has been quantitatively removed by contacting the fermentation broth with an ion-exchange resin, contains the APF- active substance originally present in the fermentation broth. This eflluent broth'can then be concentrated to produce directly a fermentation residue, free of streptomycin and non-toxic to chicks, which can be used, without further treatment, for the enrichment of animal feeds deficient in APF. Alternatively, this effluent broth can be treated with an adsorbent such as fullers earth or activated charcoal, thereby adsorbing ,APF-active substances. This adsorbate, after drying, is suitable as a feed supplement for providing APF. If a more concentrated feed. supplement, free of adsorbentis desired, the adsorbat'e can be eluted with a" suitable solvent and the eluate evaporated. to dryness toproduce a concentrated source of growth factors adapted for "the enrichment'of APF deficient animal feeds. 1 i

As previously noted, we especially prefer to prepare the novel APF concentrates utilizing a strain o'f-Fungi which synthesizes vitamin B12. The fermentation, broths obtained by fermenting aqueous nutrient mediums with these vitamin B12-synthesizing strains can be treated according to the procedurespreviously described to produce APF-active concentrates containing Fungi-synthesized vitamin B12. Where the vitamin B12-synthesizing Fungi produces an antibiotic or a material toxic to chicks the auxiliary product may be separated to produce an APF active fermentation residue containing vitamin B12 which can be further processed if desired to produce concentrates rich in the vitamin B12 component. Alternatively, the effluent broth can be treated with an adsorbent such as fullers earth and the adsorbate dried to produce a more concentrated APF active product containing vitamin B12.

Where a product of extremely high APF activity is desired or where it is desired to isolate pure vitamin B12, the fermented broth obtained by fermenting an aqueous nutrient medium with a vitamin B12-synthesizing strain of Fungi may be first treated withan adsorbent material such as activated charcoal which produces an adsorbate containing the vitamin B12 present in the broth, together with various contaminants. (If streptomycin or grisein is present, the antibiotic is adsorbed along with the vitamin B12 and, in the case of streptomycin-containing broths, it may be preferred first to treat the broth with an ion-exchange resin to adsorb streptomycin; this is not essential, however, since the subsequent purification treatment separates the vitamin B12 from othermaterials in the broth, including the antibiotic substance.) The adsorbate is then eluted with a suitable solvent such as an aqueous solution of pyridine or an alkyl substituted pyridine, and the eluate evaporated thereby producing an elaboration product which contains the vitamin B12, together with some of the contaminants, such as grisein or streptomycin, which may have been present in the original broth. This procedure is described in detail in U. S. Patent No. 2,505,053, issued April 25, 1950. y

We have discovered that such elaboration products can be treated according to procedures described in detail in our co-pending application, Serial No. 20,356, filed April 10, 1948. and in our U. S. Patent No. 2,563,794, issued August 7, 1951, to separate the vitamin B12 or other APF active material from the antibiotic to produce APF active substances free from grisein or streptomycin. Regarded in certain broader aspects, these procedures involve extracting with a solvent an elaboration product, said elaboration product being prepared, utilizing the procedure described in the preceding paragraph, from a broth fermented by a vitamin B12-synthesizing strain of Fungi, effecting a chromatographic fractionation by adding said solvent solution to a column of adsorbent material, and washing the column with solvent fractionally to (eilute the APF active substance from the adsorbent me- In accordance with one embodiment of our invention, said elaboration product (derivedfrom a vitamin B12- synthesizing strain of Fungi) is extracted with an extraction solvent such as water or a lower aliphatic watermiscible alcohol, preferably methyl alcohol. The selection of the extraction liquid is determined by the adsorbent material to be used in the chromatographic fractionation.

Various adsorbents can beutilized at various stages in the practice of this invention, including activated carbon, activated alumina, and the like. Activated alumina is the preferred adsorbent in the chromatograph step or steps. Aqueous extractions are employed when chromatograph columns containing activated carbon are used and lower aliphatic water-miscible alcoholextractions are used for activated alumina columns. The column is preferably Wet packed, by filling with the adsorbent and the solvent used in the extraction and then allowing the solvent to drain out until its level'reaches the top of the adsorbent material in the column.- The adsorbent is conveniently supported on a wire screen covered with a layer; of sand.

When an alumina column is employed for-the chro-' matograph, the alcoholic, preferably methanolic extract is poured on the upper surface" of the adsorbent in the column, and allowed to fiow into the adsorbent either by gravity, or under pressure, The column is then developed with additional amountsof the extraction solvent to develop in the column zones ;.of' adsorbent materialcom' oes-oz taining. vitamitr Biz in-difie1'iug" amounts and differing degrees ofipurity, and suitable fractions of: the eluate are collectedas'tlie zones emerge from.the bottom of'the column; Those-fractions containing sufficient activity as determined microbiologically or by virtue of their pink color are concentrated, either separately: or combined, to a. heavy syrup. This concentrate is then mixed with a lower aliphatic alcohol and a miscible non-solvent in which the active substance is insoluble, such as acetone is added. The red flocculent precipitate which forms is recovered to give'a solid'concentrate having extremely high vitamin Biz-activity which can be used for the treatmentof nutritional diseases and for other purposes for which vitamin B12 and its analogs are indicated. This concentrate also possesses high APP-activity and can be used'as a concentrated source of APF for the enrichment of feeds deficient in the animal protein factor.

The red flocculentprecipitate may be further purified by'dissolving it in a-so1ventfor vitamin B12, such as methyl or ethyl alcohol or water, and precipitating. it with a miscible non-solvent for vitamin B12, such as acetone, one or more times. Duringsuch treatment, the solution, if not'clear before adding'the precipitant, may be filteredto remove insoluble impurities. The number of such precipitationsand treatments desirable varies with different batches. A guide. tothe number of useful precipitationsis absence of brownish or yellowish color in the supernatant liquor after precipitation. In some cases, after several. suchtreatmentsvitamin B12 may be crystallized from a water solutionby adding acetone to the point of incipient turbidity and allowing red needlelike crystals to form.

However, when a crystalline product or a high degree of purification .is wanted, it is-oftendesirable to subject the product obtained from the first chromatographic fractionationteither before orafter oneor more of the abovementioned.precipitation steps) to asecond chromatographic fractionation: This. isaccomplished by dissolving the amorphous;product in alower aliphatic alcohol. The solution can be. addedto a second column of activatedalumina. and .themore. active fractions of eluate as determined-abovecan'be collected and concentrated to dryness; The dry residue may' be dissolved in methyl alcohol and acetone added to the .solution'causing a red precipitate of vitaminwBiz to: separate. This precipitate may be; dissolvedin water. and acetone added to the solutioncausingtred crystals to separate. Thecrystals mayberedissolved in water and precipitated with acetone several times to remove any. impurities that still remain.

Vitamin B12, obtainedima substantially pure state in accordance with our. invention, is a red compound having an. approximatev composition typified by the following analysis made on. two samples dried in vacuo for two hoursat 100? C.; C, 56.35,.5611; H, 6.72, 6.72; N, 14.51,.1416; P, 2.24, 2.27; Co, 4.42, 4.58.

Substantially all of the differences between the sum of the percentages given and. l%. is believed to be oxygen and no. other. metals are. indicated to be present by spectrum analysis; Based. on the foregoing. analysis, vitamin B12 has the approximate empirical formula of about C61-64H8692N14013PCO. Vitamin B12 is substantially soluble. in .water, methyl alcohol, .ethyl alcohol and phenol and substantially insoluble in acetone, ether and chloroform. Vitamin B12 has an apparent partition coefficient of about 1.46. in the system: 75% toluene-25% orthocresol: water. It has been found that vitamin B12 has a partition coefficient of 1 about 1.2 in the system water/bcnzyl alcohol.

Vitamin B12 crystallizesfrom suitable solutions such as aqueous acetone as red crystals having the following crystallographic properties: refractive indices or, 1.619; ,6, 1.649; 7, 1.659. Vitamin B12 crystals have no definite melting point but-darken at about 210-220 C.

The absorption spectrunr of vitamin B12 in aqueous solution is characterized by .maxima .at .2780

(E;,,, =-114.7) at 3610 A,

lta=203: andat 5500 A,

The spectrum does' notchange markedly with Change ofpH; intacid solution theintensity of. the 3610 A band decreasesby about 10%. and in alkaline solutionthere are other small changes and the fine structure becomes less marked.

The infra-red absorption spectrum was. determined with a carefully calibrated Perkin-Elmer 12A spectrometer. The following absorption maxima were observed:

Wave length in mu (IO- cm.)

3.05 S (broad) 4.62 W

S, M, and W designate strong, medium and weak absorption intensities.

The vitamin Bizeactive concentrates of this invention are useful as feed supplements, and they may also-be used for the treatment of nutritional diseases. While, in the specific: examples of this invention to be presently described, emphasis has been placed on the utilization of the concentrates as feed'supplements, such concentrates may be employed for the treatment of nutritional diseases and for other purposes for which vitamin B12 and its analogs are indicated as, for example, in the treatment of Addisonian pernicious'anemia and other macrocytic anemias. Thus, it is to be understood'that, whereas the following examples illustrate methods of carrying out the present invention, these examples are given for purposes of illustration and not-of'limitation.

EXAMPLE 1 A 6000 gal. fermentor was charged with lb. of concentrated beef extract (approximately 70% solids), 315 lb. of a tryptic digest of casein, 160 lb. of sodium chloride, 720 g. of ferrous sulfate heptahydrate, 144 g. of cobaltous nitrate hexahydrate, 20 liters of' soy bean oil, and about 3500 gal. of water. The solution was sterilized for 30 minutes at C., cooled to about 28 C., and inoculated'with' 300 gal. of a vegetative culture of a grisein, APF and vitamin B12 producing strain of Streptomyces griseus. The inoculated medium was fermented at atemperature'of about 28 C. for a period of 48 hours, and was mechanically agitated and aerated continuously with approximately 5000 cubic feet of air per hour. At the end of'the fermentation period, the whole culture broth'was acidificd to pH 3 with phosphoric. acid. and filtered. The filter cake was washed with water, and the filtrate, with-washings, was made'slightly alkaline with sodium hydroxide, and again filtered. The resulting fermented brothshad a pH of'7L8 and'an LLD assay 'of 2400 LLD units per ml.

A. Dried filtrate from S. griseus fermentation broth 100 gallons of. fermentation broth, prepared as described above, was evaporated at reduced pressure and at a temperature below 40 C. to a volume of about 6 gal The concentrate, which. had a microbiological activity of about 40,000LLD units per ml., was -.then spray-dried. The dried material contained about 3% moisture, andhad a microbiological activity. of about LLD units per mg.

B. Charcoal adsorbate from S. griseus fermentation broth.

3500 gallons of. the same fermented broth employed in A above was stirredwith about lb. of activated carbon (Norit) for. 30 minutes. The. adsorbate was removed by filtrationand washed by suspending in,.100 gal. of'water andagain filtering.

grams ofthe'moist carbon adsorbate was dried in a vacuum" oven at 45C; for approximately 20 hours,

'yielding 99 g. of dried adsorbate. The LLD activity of the dried adsorbate-was'f470 LLD units per mg.,,as calculated from the activity originally "present in the fermentation broth and the residual activity'of'the spent broth from the carbon adsorption.

C. Concentrate recovered from the eluate of the'charcoal adsorbate The remainder of the washed carbon adsorbate described under B (from the same fermentation batch), representing about 160 lb. of activated carbon, was eluted by stirring for 30 minutes with 167 gal. of 25% pyridine. The spent cake .was washed twice on the filter press; first by recycling40 gal. of 25% pyridine for 20 minutes; and then by-recycling28 gal. of 25% pyridine for 20 minutes. Two-thirds of the combined eluate and wash was evaporated at reduced pressure (vapor temperature below 35 C.) to, 24 liters. 150 ml. of the concentrated eluate, having an activity of about 250,000 LLD units/1111., was dried fromthe frozen state, to produce 14.1 g.' of producthaving a potency of 2800 LLD units/mg. i

D. Crystalline vitamin B12 I A solid concentrate prepared according to the procedure described-under B and C above, starting with 3500 gal. of fermented broth was extracted with methyl alcohol and the alcoholic, extract was 'then passed through a column containing activated alumina thereby adsorbing the vitamin B12. The column was eluted with fresh methyl alcohol and those fractions of the eluate showing most pronounced LLD activity were concentrated together. .The concentrated solution was then mixed with acetone whereupon crude vitamin B12 precipitated and was recovered by filtration. This. material was purified by reprecipitation from ethanol solution by the addition of acetone and the product further purified by crystallization from aqueous acetone to produce 106.0 mg. of crystalline vitamin B12.

The value of the foregoing concentrates and of vitamin B12 for replacing feed supplements containing animal protein factor was shown by the chick feed ing tests described below, in which the growth of chicks maintained on an APF deficient diet was compared with the growth of chicks on the same diet-supplemented by said concentrates and by vitamin B12. i

Chick feeding tests.-The chicks employed in these tests were hatched from eggs laid by hens maintained on a diet deficient in. APF, which had the following composition:

Day-old chicks were placed on a basal diet as follows:

. I .Per cent Soy bean meal 70.0 Commercial dextrose-.. 7.9 Cellulose 5.0 Wheat germ oil 4.5 Calcium gluconate 2.5 Glycine 2.0 DL-methionine 0.9 CaCOa 1.5 KzHPO4 1.612 KH2PO4 1.0. NaCl 0.838 MgSO4-7H2O j ;51 CaHPO4-2H2O 0.375 Ferric citrate 0.138. MnSO4-4HzO 0.025 KI u 1 0.004 CuSO4-5HzO 0.0015 ZnClz 0.00125 L-arginine 0.50 L-cysteine l 0.20 Choline v Inositol p-Aminobenzoic acid Niacin Ca pantothenate Riboflavin Thiamin 0 002 Pyridoxine Pteroylglutamic acid ..0.0004 Menadione u 0.0004 Biotin 0.00004 A '& D 0.10

"After 5 dayson the "above basalfdie t, groups-cf] i chickseach,- balanced in regard to body weight,. were selected and given the foregoing basal; diets supple: mented as indicated in-the following table: 4

The rates of growth of these chicks are summarized in the following table: 3

Increment Chick Age in over Gain Days 5 8 10 12 15 17 on C-1068 17 Days of Age Diet:

C1068-.- 49. 9 57. 3 69. 7 67. 6 66. 2 66. 5 C-1093 49. 9 58. 7 67.0 79. 3 107. 3 125. 8 59. 3 04094... 49. 9 58. 9 05.9 77. 7 '92. 4 117. 8' 51. 3 C1095 49. 9 60. 4 67. 3 82. 5 108. 7 126. 7 60. 2 G1096 49. 9 58. 6 67. 9 83.1 102. 6 122. 6 56.1

These tests clearly establish that the growth of test chicks is markedly increased, as compared with chicks fed on an APF deficient basal diet, by feeding the test chicks said basal diet enriched withjany meet the above concentrates, or vitamin B12.-- Moreover each of .th ncon centrate's (C-1094, *C-l-095 and C1096) added' in an amount not greater than 1.0% ,of the weight of the ;APF deficient chick feed have produced by a maximal growth in chicks comparable with that obtained by feeding chicks said feed; enriched with 30 mg. of vitamin B12 per kg. (i. e. 0.000003% vitamin B12).

" EXAMPLE2 A 6000 gallon fermentor was charged with lb. of beef extract concentrate, 315 lb. of a tryptic digest of casein, lb. of sodium chloride, 20 liters of antifoam agent (soy bean oil), arid 3500 gallons of water. The

3 medium was sterilized, inoculated, fermented, acidified,

filtered, and neutralized as described in Example 1.

The neutral filtered fermentation broth (approximately 4000 gallons) was stirred with 96 lb. of activated carbon for 30 minutes to adsorb the LLD active constituents. The adsorbate was removed by filtration and washed by suspending in about 100 gallons of water and again filters ing. The washed adsorbate was eluted by stirring for 30 minutes with 95 gallons of 25% alpha-picoline, .and the eluate recoveredflby filtration; The 'filter' ,cake was a washed on the filter press by recycling 30 gallons of 25 alpha-picoline for 30 minutes. The combined eluate and wash was evaporated under reduced pressure (vapor tern" perature below 35 C.) to a volume of 6 gallons and diluted with 60 gallons of methanol. Inert material, which precipitated, was removed, and the crude LLD active constitutents-were precipitated by adding the meth. anol solutionto gallons of ether. The precipitate was recovered by filtration, washed with ether, and dried in a vacuum oven at a temperature below 30 C. to produce 860 g. of dried product; microbiological potency 1500 LLD units/mg.

The value of this materialas a source of APE as compared with crude liver extract and with crystalline vitamin B12 was determined biologically using chick feed;

, ing tests'as described below.

aj'rosjaoa Amount of Supple- N r men OL-O Diet No. Supplement Percent Chicks of Total Diet None 7 o... 7 Crude Li rExtra 7 04059 S. ariseus Fermentation Concen- 0.0227 7 mite-Prepared Above.

(2-1057 Crystalline Vitamin Bn 0. 000003 The rates of growth of the chicks On these diets are summarized below:

MEAN BODY WEIGHT IN GRAMS Increment Chick Agev in Over Gain Days 5 8 10 12 15 18 on O-1014 18 DaysotAge 60.1 66.0 77.9 93.9- 113.0 }(l14;8) aver- 59.7 66.9 78.4 97.1 116.7 age. 60.2 68.3 83.5 105.7 133.2 .4 62.4 71.6 84.7 109.1 135.6 20.8 61.13 68.7 83.4 107.7 132.6 17.8

These testsshow that the growth of test chicks is markedly increasedas-compared with chicks fed. on APF deficient basal .diet-by feeding the test chicks said'basal diet enriched witheither an' S. griseus fermentation concentrate or vitamin B12. Thetests also-show-thatthe increased growth-isapproximately the same-as that produced by feedingtest chicks the basal-diet enriched with 1% of crude liver extract which is: one ofthe best of the previously known sources of the animal protein Tap water to 100%.

This medium. Was prepared, sterilized, cooled, and inoculated with 10% by volume.of-a.vegetative culture ofia streptomycin, APE and vitamin'Biz producing. strain of Streptomyces griseus. After inoculation, the inoculatedhroth was agitated'at 27 C. for 3.5. days under submerged; aerated conditions. The. fermented broth was acidified withphosphoric acid .to pH land-filtered throughdiatomaceous silica. (Supercel.).. Assay. of 'the filtered fermentation broth showed a. streptomycinactivity of'8l0'y/ml. and an LLD' activity of 2700L'LLD units/mil 60 gallons of this. filtered. fermentation broth was passed through a column containing 600gof a carboxylic acid type: ion exchange resin. (IRC 50 supplied by the Kesiuous. Productsand Chemical Co.) thereby, adsorbing the streptomycin on. the resin. The efiluent broths from which the streptomycin was thus removed were combined.

A. Charcoal adsorb/ate 7-'5 -liters ofthisefiluent broth-wastreated with' l600'g, p f-'--a 'ctivated -carbon-'-(Norit A) whereby the carbon ad 16 sorbed 75% of the.LLD-activesubstances originally; present in thetbroth.

The carbon adsorb'atc: was washed and. dried. The dry product contained approximately 80,000 LLD units/ gram and'can be used in the same'way as the charcoal adsorbate described: ixrExample l for the enrichment ofanimal feeds deficient in APP.

B. Fuller's earth adsorbate 40 liters of efiluent broth, prepared substantially as described'above (but assayingSOO LLD units perml.), were adjusted to pH 2.5 with phosphoric acid, and the resulting broth-was treated with 600 g. of fullers earth (0. K. Brand). 'Ihe fullers earth adsorbate, which contained of the LLD- activity originally present in the broth, was washed and'dried-to produce a final dry productcontaining 30,000 LLD units/gram. This product was evaluated utilizing-the chick test set forth on'pages 12'to l4 hereinabove. The APF activity per gram of the fullers earth adsorbate was found to be approximately equivalent to thatshown by 1.5 micrograms of vitamin B12 (i. e. ILS'y/gram).

C. Spray-dried concentrate An 80 liter portion of: the same effluent broth used .as thestarting material in part ATof the present example, was evaporatedto a volume. of 12 liters. A-portion of this concentrated. solution. (5740 ml.) was spray-dried toproduce .335 g. of dry product having a microbiological potency of 140' LLD units/mg. This product can be usedin the same. way as. the driedS. griseus broth describedin Example 1 for the enrichment of animal feeds deficient in APP.

D. Pure vitamin B12 A fermented broth was prepared,: as described inthe first two paragraphs ofthepresent example, by fermentingan-aqueous nutrient medium with a strain of Streptomyces griseus whichproduces both streptomycin and vitamin B12. The'resulting.broth;was filtered and the filtered broth-contactedw-ith a carboxylic acid type ionexchange resin, as set forth in paragraph. 3 of'the' present example, to producean'etlluent broth free of'streptomycm and containing substantially all of the vitamin B12 originally present in thefer'mented'broth. The effluent broth was'then treatedwithactivated carbon, whereby the vitamin B12 in the efiluent broth was adsorbed on the carbon. The carbon adsorbate. was eluted with aqueous pyridine thereby eluting the active material, and the pyridine eluate was evaporated to small volume. Ether-was added to the concentrated eluate thereby precipitating an active concentrate which was dried.

The dry-ether-precipitated' solid concentrate (1628 g. including 700 g. of filter aid), which had an LLD activity of 2500 units/mg, was ground to a fine powder and extracted four times by stirring for 30 minutes with 3500 ml. of methyl alcohol and'filtering. The combined methyl alcohol extracts were chromatographed on. 15 kg. of activatedalumina, and'the column washed with methyl alcohol. As soon as the redcolor characteristic of vitamin B12 appeared in the effiuent, a seriesof 3000 ml.- fractions were collected; The first five. of these, containing a total of" 673 'million LLD units, were combined and concentrated 'in vacuo at a temperature below 40 C. to ml. The impure vitamin B12 was recovered by pouring this solution into'120'ml. of'acetone; the resulting red precipitate was removed by centrifugation, washed with acetone and dried at room temperature. Further purification wasattained' by dissolving the solid in SV ml. of methyl alcohol and' adding 25ml; of acetone. The resulting precipitate was 'collected' bycentrifugation', washed with acetone; and driedythe' yieldwas' 89.8 mg.

The above redsolid was dissolved in 3 ml. of'methyl alcohol and chromatographedon'45g. of activated a1- umina. The column was washed with methyl alcohol. When the red color characteristic of vitamin Biz'appeared in the eluate, a series of-three to four ml. fractionswas collected. The'first five of-these, containing substantially" all of the red-co1or," were individually evaporated to dryness; dissolved inabout 0.3' m1. of water, and the solution treatedwith acetoneuntilslightly turbid. O'n standing for 24hours, red needle-like crystals formediin each'of 'thefive-tubes. The crystals were 'duced pressure (temperature below 35 prepared above can be used a mamecollectedand combined, washedwith acetone, and dried. Theyieldwas 14.1 mg. of crystalline vitamin Biz.

The various products herein described which possess an LLD activity of 65,000 LLD units to 11,000,000 LLD units per milligram are all suitable for the enrich- ;nent of animal feeds deficient in the animal protein actor.

EXAMPLE 4 A medium is prepared consisting of 1% of tryptic digest of casein, 0.3% beet extract concentrate, and, 10 parts per million of cobaltous nitrate hexahydrate. The pH is adjusted to about 7. Seven portions of this medium of 500 ml. each areplaced in 2-liter Erlenmeyer flasks and sterilized for 30'minutes at 115 C. Each flask is then inoculated with about 10 ml. of a vegetative culture of a selected strain of Stre'utomyces roseochromogenus, and fermented for 7 days on a rotaryshaking machine. The contentsof the flasks are then mixed and filtered.

One liter of filtered broth (about 1500 LLD units/ml.) is" evaporated under reduced pressure (temperature below 35 C.') and dried from the frozen state, to produce 6.9 g. of product having an activity of about 130 LLD units/mg, and an APF activity, according to the chick test, equivalent to 77 of vitamin B12 per gram.

Another portion of filtered broth, amounting to 2300 ml., is stirred at pH 7 for 30 minutes with 23 g. of activated carbon. The adsorbate thus obtained, is eluted twice by stirring for 30 minutes with'a mixture of 125 ml. of ethanol, 12.5 ml. of pyridine and 112.5 ml. of water. The combined eluates are evaporated under re- C.) and dried from the frozen state re roduce 2.3 g. of product, which assays 440 LLD units/mg. and shows an APF activity, as determined by the chick test, equivalent to 24'y of vitamin B per gram.

The dried fermentation residue and the carbon eluate in the same manner as the products described in Example 1 for the enrichment of foodstutts deficient in APF.

EXAMPLE A selected strain 'of Streptomyces albidoflavis culture is preparedand used for carryingout a fermentation exactly as described in Example 4'for Streptomyces roseochromogenus. p

The fermented broth is acidified to pH 3 with phosphoric acid, filtered, and the filtered broth is neutralized to about pH 7 with sodium hydroxide. 1570 ml. of neutral filtered broth (assaying I000 units/mL; 77 LLD units/mg.broth solids) is stirred for 30 minutes with 15.7 g. 'of"ac'tivated'carbon. The adsorbate, thusobtained, as recovered by filtration. The wet adsorbate is dried at 45 C. ina vacuum oven for 20'hours to produce 50.7 g. (including filter aid) of dried adsorbate. The LLD activity of the dried carbon adsorbate is about 30 unit's/mg. (as calculated from the potency of the original neutral filtered broth and of the spent broth from the carbon treatment). The APF activity of the adsorbate, by the chick test is equivalent to about 47 of vitamin B12 per gram;

Thisc'arbon adsorbate can k ner asthe products described in Example 1 for themrichment of foodstuffs deficient in APF.

. EXAMPLE-6 A fermentation medium was prepared containing the following;

Pfeifiersdriedlyeast percent 2 Cobalt nitrate ppm. as Co++ 10 Distilled water to make 100% be used in the same manif desired, pure vitamin B11 ac' 18 EXAMPLE 7 2000 gallons of fermented culture medium, prepared with a grisein-producing strain of S. griseus as described in Example 1, was treated by the following process for isolation of vitamin Biz-like substances. The whole broth was adjusted to pH 2.5 with phosphoric acid and filtered. The pH of the filtered broth was adjusted to 7.5 with sodium hydroxide, and the broth was then extracted with one-tenth its volume 'of a mixture of 40 parts of cresol and 60 parts of carbon tetrachloride. The water layer was reextracted with a mixture of- 25 parts of cresol and 75'1sartsef carbon tetrachloride. The two solvent extracts were combined, suflic'ient carbon tetrachloride was added to give "a' (icl izcresol ratio of 15:1, and'the'resulting solution was extracted with one-tenth its volume of water. The solvent layer was separated and reextracted with one-tenth its volume of water. The two rich-water extracts were combined and mixed with 1.5 volumes of benzyl alcohol and the rich water saturated with ammonium sulfate. The benzyl alcohol layer was separated and the aqueous layer was reextracted with one-tenth its volume of benzyl alcohol. The rich benzyl alcohol extracts were combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column was washed with a mixture of -1 part of methanol and 2 parts of acetone to remove the benzyl alcohol and yellow impurities. The rich material was eluted with methanol and the red solid was precipitated from the methanol solution anddried under vacuum. The presence of vitamin Biz-like material in the red precipitate was demonstrated by the totalcolor assay as outtoned in column 7 of the specification. v The amount of vitamin B12 and vitamin BIZ-like material was found, by measurement of optical density at 550 A wave length, to be equivalent to 274' mg.- of vitamin B12. From this preparation there was obtained 136 mg. of a red-colored crystalline, high- 1y active, vitamin produc't consisting of vitman B12 and vitamin B12-l1keS l IbS ta l 1 C S. This product contained, in addition to vitamin Biz-like substances, 6.7% of vitamin B.12,-3.S determined by the counter-current distribution procedure described in column 8 of this application, except that a 16-plate (instead of an 8-plate) transfer was used. Assay of this material according to the chick test as'outlin'ed' in"E xan1ple1 demonstrated that the crystalline-vitamin Biz-like substances isolated from-the fermentation medium were equivalent,in APF activity, to pure vitamin B12.

EXAMPLE 8 A medium composed of 3%" soy bean meal, 2% dextrose, 1% CaCOa, 0.5% NaCl and 0.001%

in tap water was prepared. 480' L. of medium was sterilizedat C. for /2' hour in each of two stainless steel fermentors. After cooling, the fermentor was inoculated with a neomyci-n-p'roducing strain of Streptomyees fradiae, produced as a vegetative culture in the above medium incubated on a rotary shaker. The fer] mentor was maintained at 27 C. and mechanically agitated and aerated for a period of 90 hours. Soy bean oil was 'added as needed as a defoamer. Assay for LLD factor content onfiltered fermented broth, by L. laetis assay, indicated 7100 and 8300 LLD units per ml. in the two fermentors. The contents of the two fermentors were combined, acidified, filtered, and the presence'of vitamin Biz-like material in the filtrate determined by the total color assay described elsewhere in this application. The total amount of vitamin B12 and vitamin B12 like substances, as estimated by the total color assay and'calculated as vitamin B12, was equivalent to mg. of vitamin B12 per 1000 gallons of fermented broth, The vitamin B12 and vitamin Biz-like substances were isolated from thebroth in the form of a crystalline mixture, utilizingsubstantially the same procedure as that described in Example 7. Assay of this mixture by the counter-current distribution method described in column 8 of this application showed the mixture to contain 12% of vitamin B1 per se. The vitamin B12 content of the fermented broth was thus 18 mg. vitamin Biz per 1000 gallons.v

A portion of the vitamin Bm'contained in the mixture of vitamin B12 and -vitamin Biz like substanees" was iso lated as a crystalline product and the purity verified by counter-current distribution. 5.4 mg. pure crystalline vitamin B12 was recovered per 1000 gallons of fermented broth. Concentrates of the fermentation broth obtained as described above, including the crystalline vitamin B12 isolated as indicated, can be used for the enrichment of animal feeds deficient in the animal protein factor.

Tap water to 100%.

Two 480 liter-portions of this medium were sterilized by heating at 120 C. for a period of /2 hour in each of two stainless steel fermentors. After cooling, the contents of each fermentor was inoculated with a culture of Alcaligenes faecalis, said culture having been produced by incubation in the above medium on a rotary shaker.

The contents of the fermentors were maintained at 27 C., aerated by a flow of air through the medium, and mechanically agitated for a period of 66 hours. Soybean oil was added as needed to prevent foaming of the contents. Samples of the fermented broths from the fermentors were assayed for LLD factor content, utilizing the L. lactis assay procedure hereinabove described, which showed that the broths contained 2340 and 2640 LLD units per ml. respectively.

The contents of the two fermentors were combined, heated to 120 C. for a period of 15 minutes and filtered, thereby removing culture cells. The amount of vitamin B12 and vitamin B12-like compounds in the filtered broth was estimated by the total color assay method previously herein described. The total amount of vitamin B12 and vitamin B1z-like compounds, calculated as vitamin B12, was found to be equivalent to 27.5 mg. of vitamin B12 per 1000 gallons of fermented broth. The vitamin B12 contained in the fermented broth was isolated as a crystalline product, utilizing the same procedure as that described in Example 1, and the purity of the product was verified by the counter-current distribution procedure. There was thus obtained 1.37 mg. of pure crystalline vitamin B12 per 1000 gallons of fermented broth. An additional quantity of 1.6 mg. of vitamin B12 was shown to be contained in the mother liquors utilizing the countercurrent distribution assay method described in column 8 of this application.

EXAMPLE 10 A medium was prepared containing the following:

Per cent Soybean meal Two 480 liter-portions of this medium were sterilized by heating at 120 C. for a period of /2 hour in each of two stainless steel fermentors. After cooling, each of the fermentors was inoculated with a culture of Pseudomonas lumichroma, an organism isolated from soil and characterized by its ability to oxidize lumichrome. The contents of the fermentors were maintained at a temperature of 27 C., and mechanically agitated and aerated for a period of 88 hours.

Soybean oil was added as needed as a defoamer. The contents of the fermentors were assayed for LLD factor content, utilizing the L. lactis assay method described hereinabove, which indicated values of 4000 and 7000 LLD units per ml. respectively in the two fermentors.

The contents of the two fermentors were combined, heated at 120 C. for a period of minutes, filtered to remove culture cells, and the total amount of vitamin B12 and vitamin Bm-Iike compounds present in the filtered broth was determined by the total color assay method described hereinabove. The total amount of vitamin B12 and vitamin B12-like compounds, as estimated by this total color assay method and calculated as vitamin B12, was equivalent to 8.3 mg. of vitamin B12 per 1000 gallons of fermented broth. Concentrates of the fermented broth can be used for the enrichment of animal feeds deficient in animal protein factor.

The preparation of APF-active products, utilizing Pseudomonas lumichroma, can be accomplished employing various mediums in place of the soybean meal medium disclosed hereinabove. Instead of Pseudomonas lumichroma other species of Pseudomonas, namely Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mildenbergii, Pseudomonas mucidolens and Pseudomolms chloraraphis can be used also for the preparation of APF active materials. These species of Pseudomonas were used to ferment aqueous nutrient mediums having the following compositions:

Medium A.1% dextrose, 1% N-Z-amine, 0.3% beef extract, 2 p. p. m. cobalt as CO(NO3)2.6H2O, and water to make Medium B.--1% dextrose, 8% distillers solubles, 2 ppm cobalt as Co(NOa)2.6I-I2O, and water to make 100%.

Medium C.1% dextrose, 6% soybean meal, 2 p. p. m. cobalt as CO(NO3)2.6H2O, and water to make 100%.

These mediums were prepared and subdivided into 250 ml. flasks containing 40 ml. per flask. The flasks and their contents were sterilized and each of the flasks was inoculated with a different species of Pseudomonas. After inoculation, the inoculated broths were incubated on a 220 R. P. M. shaker at 28 C. for a period of 5 days, and the activities of the fermented broths thus obtained were Each of the fermented broths thus obtained utilizing these microorganisms can be treated as described in Example 1 to produce a concentrate adapted for the enrichment of animal feeds deficient in the animal protein factor.

EXAMPLE 11 A fermentation medium was prepared containing the following:

Per cent Yeast extract 1 Dextrose 1 C0(NO3)2.6H2O 0.001

Distilled water to make 100% (3.2 liters).

The above medium was sterilized by heating at a temperature of C. for a period of one-quarter hour in a 5 liter fermentor. The sterilized medium was inoculated with a culture of Mycobacterium smegmatis, produced by incubation in the above medium. After inoculation, the inoculated broth was maintained at a temperature of 27 C. for a period of approximately 94 hours, during which time the fermenting mixture was subjected to agitation and aeration. After completion of the fermentation period, the LLD assay of the fermented broth was 4000 LLD units per ml.

The fermented broth was then autoclaved at 120 C. for a period of A to /2 hour and the culture cells were removed by filtration. The amount of vitamin B12 and vitamin Biz-like compounds in the filtered broth was estimated by processing the entire broth by the total color assay method described hereinabove. The total amount of vitamin B12 and vitamin B1z-like compounds, as estimated by this total color assay method and calculated as vitamin B12, was equivalent to mg. of vitamin B12 per 1000 gallons of broth. Concentrates of this fermented broth can be used for the enrichment of animal feeds deficient in animal protein factor.

EXAMPLE 12 A fermentation medium was prepared containing the following:

Per cent Soybean meal- 6 Dextrose 1 Co(NO3)2.6H2O 0.001 Distilled water to make 100% (3.2 liters).

: medium.was.sterilizedlbyr=lieating at a temperature of 120 Cafor a-vperiodof. approximately A hour in a 5 liter fermentor. The'steriliz'ed" medium was then inoculated with. a. culture of kseudomonast 'lu'michroma, produced by incubation in the above medium. After 'inoculatiom; the: inoculated broth waszmaintained at a temperature of 27 C. for a period of approximately 70 hours, during which time the fermentation mixture was subjected to agitation and aeration.

After completion of the fermentation period, the LLD assay of the fermented broth was 5000 LLD units per ml.

The fermented broth was then autoclaved at a temperature of 120 C. for a period of A to /2 hour and the culture cells removed by filtration. The amount of vitamin B12 and vitamin Biz-like compounds in the filtered broth was estimated by processing the entire broth by the total color assay method described hereinabove. The total amount of vitamin Biz-like compounds, as estimated by this total color assay method and calculated as vitamin B12, was equivalent to 344 mg. of vitamin B12 per 1000 gallons of fermented broth. Concentrates of this fermented broth can be used for the enrichment of animal feeds deficient in animal protein factor.

EXAMPLE 13 A fermentation medium was prepared containing the following:

Per cent Soybean meal 2 Distillers solu 0.5 Dextrose 1.0 CO(NO3)2.6H2O 0.001 Salt solution 1 0.125 Distilled water to make 100% (3.2 liters). 30

1 Solution contained 0.5 gm. KH PO 0.5 gm. MgSO 0.01 gm. NaCl, 0.01 gm. FeS0 and distilled water to 1 liter.

.01 gm, MnS0.,

This medium was sterilized by heating at 120 C. for A hour in a 5 liter fermentor. The sterilized broth was then inoculated with a culture of Alternaria alevaeca, produced by incubation of the above medium. After inoculation, the inoculated broth was maintained at a temperature of 27 C. for a period of approximately 94 hours, during which time the fermentation mixture was subjected to agitation and aeration. After completion of the fermentation period, the LLD assay of the fermented broth was 4200 LLD units per ml.

The fermented broth was then autoclaved at a temperature of 120 C. for a period of A; to /2 hour, and the culture cells were removed by filtration. The amount of vitamin B12 and vitamin Biz-like compounds in the filtered broth was estimated by processing the entire broth by the total color assay method described hereinabove. The total amount of vitamin B12 and vitamin Biz-like compounds, as estimated by this total color assay method and calculated as vitamin B12, was equivalent to 200 mg. of vitamin B12 per 1000 gallons of fermented broth. Con centrates of this fermented broth can be used for the en- ;ichment of animal feeds deficient in animal protein actor.

Various changes and modifications may be made in carrying out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are within the purview of the annexed claims, they are to be considered as part of our invention.

We claim:

1. A vitamin Biz-active composition comprising recovered elaboration products of the fermentation of a vitamin Biz-activity producing strain of Fungi selected from the class consisting of Schizomycetes, Torula, and Erem-othecium, the L. L. D. activity of said composition being at least 440 L. L. D. units per milligram and less than 11 million L. L. D. units per milligram.

2. A vitamin B12-active composition comprising recovered elaboration products of the fermentation of a vitamin Biz-activity producing strain of Fungi selected from the class consisting of Schizomycetes, Torula, and Eremothecium, the L. L. D. activity of said composition being at least 1,500 L. L. D. units per milligram and less than 11 million L. L. D. units per milligram.

3. A vitamin Biz-active composition comprising rem. E l-IP0 0.2

1 22 covered elaboration- 'products'wf the fermentation of a vitamin Biz-activity.producinggstnain' of Fungi selected from the class consisting of Schizomycetes, Torula, and Eremothecium, the L.- L. D. activity of-said composition beingat least 65 ,000-L.'L.-D-. units" per milligram-and lessthan 1 1 million L. LrD. units per milligram.

4'. A process fort-he product-ion 'of' an L. L. D.-ac'tive composition-which comprises-fermenting an aqueous nutrient medium under submerged aerated conditions by means of an L. L. D.-activity producing strain of Fungi selected from the class consisting of Schizomycetes, Torula, and Eremothecium, extracting L. L. D.-active substances therefrom, and recovering from the resulting extract an L. L. D.-active composition having an L. L. D. activity of at least 440 units per milligram.

5. A process for the production of a vitamin Biz-active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of Fungi selected from the class consisting of Schizomycetes, Torula, and Eremothecium, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 440 units per milligram.

6. A process for the production of a vitamin Biz-active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of a Schizomycetes, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 440 L. L. D. units per milligram.

7. A process for the production of a vitamin Biz-active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of an Alcaligenes, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 440 L. L. D. units per milligram.

8. A process for the production of a vitamin B12- active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of a Pseudomonas, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 440 L. L. D. units per milligram.

9. A process for the production of a vitamin Biz-active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of a Bacillus, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 440 L. L. D. units per milligram.

10. A process for the production of a vitamin Biz-active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of a Mycobacterium, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Big-active composition having an L. L. D. activity of at least 440 L. L. D. units per milligram.

11. A process for the production of a vitamin B12- active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of Fungi selected from the class consisting of Schizomycetes, Torula, and Eremothecium, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 1,500 L. L. D. units per milligram.

12. A process for the production of a vitamin B12- active composition which comprises fermenting an aqueous nutrient medium under submerged aerated conditions by means of a vitamin Biz-activity producing strain of a Schizomycetes, extracting vitamin Biz-active substances therefrom, and recovering from the resulting extract a vitamin Biz-active composition having an L. L. D. activity of at least 1,500 L. L. D. units per milligram.

(References on following page) 23 References Cited in the file of this patent UNITED STATES PATENTS Jean Dec. 12, 1939 Novak Aug. 24, 1948 Martin Dec. 20, 1949 Turner July 17, 1951 Rickes Aug. 7, 1951 24 Wood et a1 May 6, 1952 OTHER REFERENCES Le Page et a1., Jour. Biol. Chem. 162 (1946), pages 5 163-171.

Rickes et al., Science, Dec. 3, 1948, vol. 108, pages 634-5. 

1. A VITAMINE B12-ACTIVE COMPOSITION COMPRISING RECOVERED ELABORATION PRODUCTS OF THE FERMENTATION OF A VITAMIN B12-ACTIVITY PRODUCING STRAIN OF FUNGI SELECTED FROM THE CLASS CONSISTING OF SCHIZOMYCETES, TORULA, AND EREMOTHECIUM, THE L.L.D. ACTIVITY OF SAID COMPOSITION BEING AT LEAST 440 L.L.D. UNITS PER MILLIGRAM AND LESS THAN 11 MILLION L.L.D. UNITS PER MILLIGRAM.
 4. A PROCESS FOR THE PRODUCTION OF AN L.L.D.ACTIVE COMPOSITION WHICH COMPRISES FERMENTING AN AQUEOUS NUTRIENT MEDIUM UNDER SUBMERGED AERATED CONDITIONS BY MEANS OF AN L.L.D.-ACTIVITY PRODUCING STRAIN OF FUNGI SELECTED FROM THE CLASS CONSISTING OF SCHIZOMYCETES, TORULA, AND EREMOTHECIUM, EXTRACTING L.L.D.-ACTIVE SUBSTANCES THEREFROM, AND RECOVERING FROM THE RESULTING EXTRACT AN L.L.D.-ACTIVE COMPOSITION HAVING AN L.L.D. ACTIVITY OF AT LEAST 440 UNITS PER MILLIGRAM. 